Antilisterial Activity of Peptide AS-48 and Study of Changes Induced in the Cell Envelope Properties of an AS-48-Adapted Strain of Listeria monocytogenes

The peptide AS-48 is highly active on all Listeria species. It has a bactericidal and bacteriolytic mode of action on Listeria monocytogenes CECT 4032, causing depletion of the membrane electrical potential and pH gradient. The producer strain Enterococcus faecalis A-48-32, releases sufficient amounts of AS-48 into the growth medium to suppress L. monocytogenes in cocultures at enterococcus-to-listeria ratios above 1 at 37°C or above 10 at 15°C. As the temperature decreases, the bactericidal effects of AS-48 are less pronounced, but at 2.5 μg/ml it still can inhibit the growth of listeria at 6°C. AS-48 is highly active on liquid cultures, although concentrations above 0.2 μg/ml are required to avoid adaptation of listeria. AS-48-adapted cells can be selected at low (but still inhibitory) concentrations, and they can be inhibited completely by AS-48 at 0.5 μg/ml. The adaptation is lost gradually upon repeated subcultivation. AS48^ad cells are cross-resistant to nisin and show an increased resistance to muramidases. Their fatty acid composition is modified: they show a much higher proportion of branched fatty acids as well as a higher C_{15:0 An}-to-C_{17:0 An} ratio. Resistance to AS-48 is also maintained by protoplasts from AS48^ad cells. Electron microscopy observations show that the cell wall of AS48^ad cells is thicker and less dense. The structure of wild-type cells is severely modified after AS-48 treatment: the cell wall and the cytoplasmic membrane are disorganized, and the cytoplasmic content is lost. Intracytoplasmic membrane vesicles are also observed when the wild-type strain is treated with high AS-48 concentrations.     

The major allergen from birch tree pollen, Bet v 1, binds and permeabilizes membranes

The 159 residue Bet v 1 is the major allergen from birch tree pollen. Its natural function is unknown although it is capable of binding several types of physiologically relevant ligands in a centrally placed cavity in the protein structure. Here we use circular dichroism and fluorescence spectroscopy to show that Bet v 1 binds to DOPC and DOPG phospholipid vesicles in a pH-dependent manner. Binding is facilitated by low pH, negatively charged phospholipids, and high vesicle curvature, indicating that electrostatic interactions and vesicle surface defects are important parameters for binding. Binding is accompanied by major structural rearrangements, involving an increase in alpha-helical structure and a decrease in beta-structure. A bilayer structure per se is not a prerequisite for these rearrangements, since they also occur in the presence of the micelle-forming lysophospholipids lysoMPC and lysoMPG. Two major bound states (A and B) with distinct secondary structure compositions were identified, which predominate in the pH ranges approximately 9.5-6.5 and approximately 5-2.5, respectively. Despite the high content of secondary structure, the A- and B-states are partially unfolded as they unfold noncooperatively in CD thermal scans, in contrast to the native state. In addition, the B-state (but not the A-state) shows intermediate proteolysis-resistance and is able to induce complete leakage of calcein from the vesicles, indicating that this state is partially inserted into and significantly perturbs the bilayer structure. We conclude that Bet v 1 is a membrane binding protein, highlighting a possible biological function of this protein.     

Atypical polyproline recognition by the CMS N-terminal Src homology 3 domain

The CIN85/CMS (human homologs of mouse SH3KBP1/CD2AP) family of endocytic adaptor proteins has the ability to engage multiple effectors and couple cargo trafficking with the cytoskeleton. CIN85 and CMS (Cas ligand with multiple Src homology 3 (SH3) domains) facilitate the formation of large multiprotein complexes required for an efficient internalization of cell surface receptors. It has recently been shown that c-Cbl/Cbl-b could mediate the formation of a ternary complex between one c-Cbl/Cbl-b molecule and two SH3 domains of CIN85, important for the ability of Cbl to promote epidermal growth factor receptor down-regulation. To further investigate whether multimerization is conserved within the family of adaptor proteins, we have solved the crystal structures of the CMS N-terminal SH3 domain-forming complexes with Cbl-b- and CD2-derived peptides. Together with biochemical evidence, the structures support the notion that, despite clear differences in the interaction surface, both Cbl-b and CD2 can mediate multimerization of N-terminal CMS SH3 domains. Detailed analyses on the interacting surfaces also provide the basis for a differential Cbl-b molecular recognition of CMS and CIN85.     

A strain of Holmes' ribgrass virus occurring in Yugoslavia

A virus having 300 nm long rod-shaped particles was isolated fromPlantago media L. in Yugoslavia. The virus was transmitted to 15 species of host plants the symptoms of which are described in detail. The symptoms corresponded to those that appeared after infection by the original Holmes' ribgrass virus (HRV). The investigated virus was compared both with the common strain of tobacco mosaic and the original Holmes' ribgrass viruses by means of serological tests. The agar double-diffusion tests showed that it is closely related to HRV and remotely related to the common strain of TMV. On the basis of these results we concluded that this virus represented a strain of the HRV. The investigations of the cell inclusions showed that our virus produced rounded plates instead of hexagonal prisms. Electron micrographs of ultrathin sectioned material demonstrated that these plates were formed by virus particles lying perpendicularly to the layers of the plates. The presence of plates also points to the fact that the investigated virus belongs to the HRV.     

Multiple phosphorylation of rat liver 3-hydroxy 3-methylglutaryl coenzyme a reductase

Rat liver homogeneous ³²P-labeled hydroxy methylglutaryl coenzyme A reductase, was treated independently with CNBr and trypsin and the resulting [³²P]phosphopeptides were analyzed by disc gel electrophoresis. CNBr treatment produced only one ³²P-fragment of Mr 18,000. The time course of trypsin hydrolysis initially showed the appearance of some phosphopeptides, which were lately converted in two phosphopeptides of low Mr. These results provide direct support for the concept that hydroxy methyl glutaryl coenzyme A reductase kinase solubilized from microsomes phosphorylates only two sites or set of sites in the reductase molecule.     

Evaluation of matrix-assisted laser desorption ionization-time of flight whole cell profiles for assessing the cultivable diversity of aerobic and moderately halophilic prokaryotes thriving in solar saltern sediments

The moderately halophilic, cultivable fraction of prokaryotes thriving in hypersaline sediments of a solar saltern in Mallorca, Spain, has been studied by means of different cultivation media. A set of 374 isolates retrieved with six different culture conditions was screened, using whole-cell MALDI-TOF MS analysis to classify them into 25 phenotypic clusters at 52% similarity. The phylogenetic inference, made from comparative sequence analyses of the 16S rRNA genes of selected strains, indicated that each phenotypic cluster was comprised of a genealogically homogeneous set of strains. DNA-DNA hybridization (DDH) results among selected strains confirmed that each MALDI-TOF cluster encompassed members of the same species. On the other hand, the intra-cluster diversity, measured by several RAPD (Random Amplified Polymorphic DNA) amplifications, indicated that the clusters corresponded to several populations of the same phylogenetic unit coexisting in the same environment. The results encourage the use of MALDI-TOF MS for further exhaustive studies of the cultivable diversity of hypersaline environments.     

Oxidative Stress Induced in Sunflower Seedling Roots by Aqueous Dry Olive-Mill Residues

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O₂·^- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O₂·^- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O₂·^- and H₂O₂ accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H₂O₂ content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.     

The vasodepressor effect of androgens in pithed rats: potential role of calcium channels

Estrogens induce vasodilatation and/or hypotension in several experimental models, probably by a blockade of calcium currents. However, very little is known about the potential cardiovascular effects of androgens. We have previously shown that 5 beta-reduced androgens are more potent vasorelaxants than their precursors (delta 4-3 keto), 5-reduced progestins and 17beta-estradiol. The present study set out to investigate if this vasorelaxant effect of 5-reduced androgens is operative in vivo in the analysis of the potential vasodepressor effect of these compounds in vagosympathectomized, pithed rats. After increasing diastolic blood pressure (DBP) by a continuous infusion of norepinephrine (0.059 micromol x kg(-1)min(-1)), i.v. bolus injections of 3 alpha-hydroxy-5 beta-androstan-17-one (etiocholanolone), 5 beta-dihydrotestosterone (5 beta-DHT), and its isomer 5 alpha-dihydrotestosterone (5 alpha-DHT) (5-25 micromol x kg(-1) each) produced, separately, dose-dependent vasodepressor responses. These responses were biphasic: an immediate fall in DBP (reaching the nadir within 1.7 min) was followed by a further slow decrease that reached a maximum between 80 and 100 min after steroid administration. The order of potency of androgens in decreasing DBP was: 5 beta-DHT>5 alpha-DHT=etiocholanolone for the short-lasting response and 5 alpha-DHT>5 beta-DHT>or=etiocholanolone for the longer lasting response. Importantly, the same doses of these compounds produced no significant changes in heart rate. Moreover, 5 beta-DHT significantly antagonized the vasopressor responses to methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) with a blocking profile similar to that of nifedipine (NIF). This finding suggests that a blockade of voltage-operated calcium channels may be involved in androgen-induced hypotension.